U.S. patent application number 14/166945 was filed with the patent office on 2014-08-21 for vaccine composition for mucosal administration.
The applicant listed for this patent is NITTO DENKO CORPORATION. Invention is credited to Daisuke ASARI, Mitsuhiko HORI, Wenjing LI, Yoshiki MAEDA, Kyohei MATSUSHITA, Arimichi OKAZAKI, Katsuyuki OKUBO, Takuya SHISHIDO, Haruo SUGIYAMA.
Application Number | 20140234377 14/166945 |
Document ID | / |
Family ID | 50028731 |
Filed Date | 2014-08-21 |
United States Patent
Application |
20140234377 |
Kind Code |
A1 |
OKAZAKI; Arimichi ; et
al. |
August 21, 2014 |
VACCINE COMPOSITION FOR MUCOSAL ADMINISTRATION
Abstract
The present invention provides a vaccine composition which
comprises a cellular immunity induction promoter universally usable
against various antigens in cellular immunity induction by mucosal
administration of the antigen and exerts a high cellular immunity
inducing effect by mucosal administration. The present invention
provides a vaccine composition for mucosal administration to induce
cellular immunity, comprising: (i) an antigen; and (ii) one or more
cellular immunity induction promoters selected from the group
consisting of a TLR ligand, a cyclic dinucleotide, a helper peptide
and an immunomodulatory small molecule drug.
Inventors: |
OKAZAKI; Arimichi; (Osaka,
JP) ; MATSUSHITA; Kyohei; (Osaka, JP) ; ASARI;
Daisuke; (Osaka, JP) ; SHISHIDO; Takuya;
(Osaka, JP) ; MAEDA; Yoshiki; (Osaka, JP) ;
OKUBO; Katsuyuki; (Osaka, JP) ; LI; Wenjing;
(Osaka, JP) ; HORI; Mitsuhiko; (Osaka, JP)
; SUGIYAMA; Haruo; (Osaka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NITTO DENKO CORPORATION |
OSAKA |
|
JP |
|
|
Family ID: |
50028731 |
Appl. No.: |
14/166945 |
Filed: |
January 29, 2014 |
Current U.S.
Class: |
424/277.1 |
Current CPC
Class: |
A61K 39/00115 20180801;
A61K 39/001106 20180801; A61K 2039/572 20130101; A61P 31/12
20180101; A61K 39/001186 20180801; A61P 37/00 20180101; A61P 37/04
20180101; A61P 35/00 20180101; A61K 2039/55516 20130101; A61K
2039/55511 20130101; A61K 2039/55566 20130101; A61K 2039/80
20180801; A61K 2039/55561 20130101; A61K 2039/55583 20130101; A61K
39/0011 20130101; A61K 39/39 20130101; A61K 2039/542 20130101; A61K
39/00117 20180801 |
Class at
Publication: |
424/277.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 5, 2013 |
JP |
2013-020910 |
Claims
1. A method for inducing cellular immunity in a subject, which
comprises mucosally administering a cancer vaccine composition
comprising: (i) an antigen; and (ii) at least one more cellular
immunity induction prompter selected from the group consisting of a
TLR ligand, a cyclic dinucleotide, a helper peptide and an
immunomodulatory small molecule drug to the subject.
2. The method according to claim 1, wherein the cellular immunity
induction prompter is a helper peptide.
3. The method according to claim 1, wherein the cellular immunity
induction prompter is a combination of a helper peptide and at
least one substance selected from the group consisting of a TLR
ligand, a cyclic dinucleotide and an immunomodulatory small
molecule drug.
4. The method according to claim 1, wherein the antigen is a
peptide selected from the group consisting of survivin 2B peptide
and/or modified survivin-2B peptide, GPC3 peptide and/or modified
GPC3 peptide, HER2/neu_A24 peptide and/or modified HER2/neu_A24
peptide, MAGE3_A24 peptide and/or modified MAGE3_A24 peptide,
IPEP87 peptide and/or modified IPEP87 peptide, PR1 peptide and/or
modified PR1 peptide, HER2/neu_A02 peptide and/or modified
HER2/neu_A02 peptide, MAGE3_A02 peptide and/or modified MAGE3_A02
peptide, HBVenv peptide and/or modified HBVenv peptide, and MUC1
peptide and/or modified MUC1 peptide.
5. The method according to claim 1, wherein the method is for
treating a cancer.
6. The method according to claim 1, wherein the method is for
treating a viral disease.
Description
TECHNICAL FIELD
[0001] The present invention relates to a vaccine composition for
mucosal administration. More particularly, it relates to a vaccine
composition for mucosal administration to induce cellular immunity,
comprising (i) an antigen; and (ii) one or more cellular immunity
induction promoters.
BACKGROUND ART
[0002] Vaccines are widely used in order to induce immunity into
the subject and include those for administering pathogens such as
microorganisms or viruses, or a part thereof. There is a cancer
vaccine for allowing a cellular immunity mechanism to recognize a
cancer cell specific antigen and inducing a specific attack of the
immune system to cancer cells, which is used as one measure for
treating a cancer.
[0003] In usual, the invasion of microorganisms and viruses into
the bio-body is prevented by skin due to the size thereof, and it
is necessary to invasively administrate a vaccine into the
bio-body. Accordingly, injections are usually used in order to
provide immunity. Injections, however, have problems of pain, fear,
an injection scar, and a needle mark and scarring thereof, and have
further problems that only a medical worker is allowed to perform
such administration; it is technically difficult to perform an
intradermal injection having a high immune effect; there is a risk
such as an infection accident caused by needlestick by a medical
worker; patients are forced to go repeatedly to the hospital when
repeated injection is required; and it causes medical wastes such
as injection needle which is required to be disposed by a special
method. Thus, injection is not necessarily an optimal route of
administration.
[0004] Subcutaneous injection or intradermal injection is most
generally used as the route of administration of a vaccine, but in
addition to them, various routes of administration have been tried
to induce immunity, for example, transdermal administration (Patent
Document 1 and Non-Patent Document 1), buccal administration,
transnasal administration, and sublingual administration
(Non-Patent Document 2, Patent Document 2 and Patent Document
3).
[0005] In order to provide immunity by injection, it is usually
used an adjuvant. For example, aluminum salts such as aluminum
hydroxide, aluminum phosphate and aluminum chloride, and emulsions
including squalene such as MF59 and AS03 are practically used as an
adjuvant, and in addition to them, flagellum components, nucleic
acids, cytokines, cationic polymers and polypeptides are widely
studied as an adjuvant. With respect to an adjuvant to be used for
other route than injection such as transdermal administration or
transmucosal administration to provide immunity, it has also been
studied to use a substance such as aluminum salts (e.g. aluminum
hydroxide, aluminum phosphate and aluminum chloride), and toxins
(e.g. cholera toxin and heat-labile E. coli toxin), but they have
not yet been put into practical use. Most of them are used as an
adjuvant for inducing humoral immunity by producing antibodies to
prevent infection from viruses or bacteria. On the other hand, as
for only cellular immunity induction, a Freund adjuvant, Montanide,
GM-CSF, IL-2, IL-12 and IFN-.gamma. have been studied as an
adjuvant for injection, but they have still not yet been put into
practical use. Besides, in the route of transdermal administration
or mucosal administration, there are only a few reports about
toxins such as cholera toxin and heat-labile E. coli toxin, and
nucleic acids.
LIST OF DOCUMENTS
[0006] [Patent Document 1] US-A-2008/0193487 [0007] [Patent
Document 2] JP-A-2002-531415 [0008] [Patent Document 3]
US-A-2008/0112974 [0009] [Patent Document 4] JP-A-7-505883 [0010]
[Patent Document 5] JP-A-2007-529531 [0011] [Non-Patent Document 1]
Hosoi Akihiro et al., Cancer Research, 68, 3941-3949 (2008) [0012]
[Non-Patent Document 2] Zhengrong Cui et al., Pharmaceutical
Research, Vol. 19, No. 7, 947-953 (2002)
SUMMARY OF THE INVENTION
[0013] Mucosal administration has been thought as one measure for
solving various problems regarding injection. However, there is
little report as to a promoter which can induce cellular immunity
by mucosal administration of an antigen. In particular in a case of
using a peptide as the antigen, there are no report of cellular
immunity induction by mucosal administration. Thus, a sufficient
cellular immunity induction effect cannot be obtained in mucosal
administration, contrary to the route of injection.
[0014] An object of the present invention is to provide a vaccine
composition which comprises the cellular immunity induction
promoter and is highly effective for cellular immunity induction by
mucosal administration.
[0015] The present invention provides a vaccine composition which
comprises a cellular immunity induction promoter universally usable
against various antigens in immunity induction by mucosal
administration of an antigen and exerts a high cellular immunity
inducing effect in mucosal administration. In one aspect of the
present invention, the cellular immunity induction caused by
mucosal administration of an antigen is potentiated by using a
specific cellular immunity induction promoter together with the
antigen.
[0016] Specifically, when one or more cellular immunity induction
promoters selected from the group consisting of a TLR ligand, a
cyclic dinucleotide, a helper peptide and an immunomodulatory small
molecule drug are used in a vaccine composition for mucosal
administration, a high cellular immunity inducing effect can be
obtained in mucosal administration.
[0017] The present invention provides the aspects listed below.
(1) A vaccine composition for mucosal administration to induce
cellular immunity, comprising (i) an antigen; and (ii) one or more
cellular immunity induction promoters selected from the group
consisting of a TLR ligand, a cyclic dinucleotide, a helper peptide
and an immunomodulatory small molecule drug; (2) The vaccine
composition according to (1), wherein the cellular immunity
induction promoter is a TLR ligand; (3) The vaccine composition
according to (1), wherein the cellular immunity induction promoter
is a cyclic dinucleotide; (4) The vaccine composition according to
(1), wherein the cellular immunity induction promoter is an
immunomodulatory small molecule drug; (5) The vaccine composition
according to (1), wherein the cellular immunity induction promoter
is a helper peptide; (6) The vaccine composition according to (1),
wherein the cellular immunity induction promoter is a combination
of one or more substances selected from the group consisting of a
TLR ligand, a cyclic dinucleotide and an immunomodulatory small
molecule drug, and a helper peptide; and (7) The vaccine
composition according to any one of (1) to (6), wherein the antigen
is a peptide selected from the group consisting of survivin 2B
peptide and/or modified survivin-2B peptide, GPC3 peptide and/or
modified GPC3 peptide, HER2/neu_A24 peptide and/or modified
HER2/neu_A24 peptide, MAGE3_A24 peptide and/or modified MAGE3_A24
peptide, IPEP87 peptide and/or modified IPEP87 peptide, PR1 peptide
and/or modified PR1 peptide, HER2/neu_A02 peptide and/or modified
HER2/neu_A02 peptide, MAGE3_A02 peptide and/or modified MAGE3_A02
peptide, HBVenv peptide and/or modified HBVenv peptide, and MUC1
peptide and/or modified MUC1 peptide.
[0018] In another aspect, the vaccine composition of the present
invention can be used for the treatment or prevention of diseases.
Therefore, the present invention also provides the embodiments
listed below.
(8) A method for treating or preventing a cancer comprising
mucosally administrating to a subject a therapeutically effective
amount of (i) a cancer antigen, and (ii) one or more cellular
immunity induction promoters selected from the group consisting of
a TLR ligand, a cyclic dinucleotide, a helper peptide and an
immunomodulatory small molecule drug; (9) The method according to
(8), wherein the cancer antigen is a cancer antigen peptide
selected from the group consisting of survivin 2B peptide and/or
modified survivin-2B peptide, GPC3 peptide and/or modified GPC3
peptide, HER2/neu_A24 peptide and/or modified HER2/neu_A24 peptide,
MAGE3_A24 peptide and/or modified MAGE3_A24 peptide, PR1 peptide
and/or modified PR1 peptide, HER2/neu_A02 peptide and/or modified
HER2/neu_A02 peptide, MAGE3_A02 peptide and/or modified MAGE3_A02
peptide, and MUC1 peptide and/or modified MUC1 peptide; (10) A
method for treating or preventing a viral disease comprising
mucosally administrating to a subject a therapeutically effective
amount of (i) a virus antigen, and (ii) one or more cellular
immunity induction promoters selected from the group consisting of
a TLR ligand, a cyclic dinucleotide, a helper peptide and an
immunomodulatory small molecule drug; and (11) The method according
to (10), wherein the virus antigen is a peptide selected from the
group consisting of IPEP87 peptide and/or modified IPEP87 peptide,
and HBVenv peptide and/or modified HBVenv peptide.
[0019] In another aspect, the present invention provides a TLR
ligand, a cyclic dinucleotide, a helper peptide, an
immunomodulatory small molecule drug, or a mixture of two or more
thereof, for use as a cellular immunity induction promoter for
mucosal administration against an antigen. The present invention
also provides the following aspect:
(12) A TLR ligand, a cyclic dinucleotide, a helper peptide, an
immunomodulatory small molecule drug, or a combination of two or
more thereof, for use as a cellular immunity induction promoter in
cellular immunity induction by mucosal administration of an
antigen.
[0020] The present invention also provides the following
embodiments:
(13) A method of inducing cellular immunity, comprising mucosally
administering to a subject (i) an antigen and (ii) one or more
cellular immunity induction promoters selected from the group
consisting of TLR ligand, a cyclic dinucleotide, a helper peptide
and an immunomodulatory small molecule drug; (14) TLR ligand,
cyclic dinucleotide, helper peptide, immunomodulatory small
molecule drug or a combination of two or more kinds of them, for
use in promoting the induction of cellular immunity by the mucosal
administration of an antigen; (15) A combination of (i) an antigen
and (ii) one or more cellular immunity induction promoters selected
from the group consisting of TLR ligand, a cyclic dinucleotide, a
helper peptide and an immunomodulatory small molecule drug, for use
in inducing cellular immunity by the mucosal administration of an
antigen; (16) A combination of (i) a cancer antigen and (ii) one or
more cellular immunity induction promoters selected from the group
consisting of TLR ligand, a cyclic dinucleotide, a helper peptide
and an immunomodulatory small molecule drug for use in treating or
preventing a cancer, wherein the combination is mucosally
administered to a subject; (17) A combination of (i) a virus
antigen and (ii) one or more cellular immunity induction promoters
selected from the group consisting of TLR ligand, a cyclic
dinucleotide, a helper peptide and an immunomodulatory small
molecule drug for use in treating or preventing a viral disease,
wherein the combination is mucosally administered to a subject;
(18) Use of (i) an antigen and (ii) one or more cellular immunity
induction promoters selected from the group consisting of TLR
ligand, a cyclic dinucleotide, a helper peptide and an
immunomodulatory small molecule drug, for the manufacture of a
vaccine composition for mucosal administration intended for the
induction of cellular immunity; (19) Use of (i) a cancer antigen
and (ii) one or more cellular immunity induction promoters selected
from the group consisting of TLR ligand, a cyclic dinucleotide, a
helper peptide and an immunomodulatory small molecule drug, for the
manufacture of a vaccine composition for mucosal administration
intended for the treatment or prevention of a cancer; and (20) Use
of (i) a virus antigen and (ii) one or more cellular immunity
induction promoters selected from the group consisting of TLR
ligand, a cyclic dinucleotide, a helper peptide and an
immunomodulatory small molecule drug, for the manufacture of a
vaccine composition for mucosal administration intended for the
treatment or prevention of a viral disease.
[0021] The vaccine composition of the present invention can be
mucosally administered (in particular, transnasally and through
oral mucosal membranes including a sublingual mucous membrane), and
thus it has advantages of excellent compliance, for example,
noninvasive administration, painlessness, and release from fear of
injection, and has further advantages that the composition can be
administered by a patient himself/herself because of ease of
administration; a risk of an infection accident caused by
needlestick by a medical worker can be avoided; the frequency of
hospital visit when repeated administrations are performed can be
reduced, which contributes to improve the life quality of the
patient; and further medical wastes such as an injection needle is
not generated. In addition, the vaccine composition of the present
invention has also an advantage that the effect as the vaccine is
remarkably improved as compared with the case of single
administration of an antigen. Furthermore, the vaccine composition
of the present invention has also an advantage that the mucosal
administration thereof can induce stronger immunity than the
injection administration.
DETAILED DESCRIPTION OF THE INVENTION
[0022] In order to more easily understand the present invention,
the terms as used herein are defined below. The terms not defined
herein have meanings generally understood by those skilled in the
art, particularly in the fields of medical science, pharmacy,
immunology, cell biology, biochemistry, and polymer chemistry,
unless the context requires otherwise.
I. DEFINITION
[0023] The term "antigen" as used herein means any substance
capable of inducing an immune response, for example, proteins and
peptides. In the mucosal administration, in which the antigen shall
be permeable through the mucous membrane, it is preferable to use
an antigen having a small molecular weight, for example, a peptide
having about 8 to about 12 amino acids. Examples of peptide
antigens to be used in the present invention include the following
peptides: survivin-2B peptide, GPC3 peptide, HER2/neu_A24 peptide,
MAGE3_A24 peptide, IPEP87 peptide, PR1 peptide, HER2/neu_A02
peptide, MAGE3.sup.--A02 peptide, HBVenv peptide, HER2/neu E75
peptide, and MUC1 peptide. In one embodiment, one or more peptides
selected from the group consisting of HER2/neu E75 peptide for
cancer vaccine applications, modified HER2/neu E75 peptide for
cancer vaccine applications, WT1 peptide for cancer vaccine
applications, and modified WT1 peptide for cancer vaccine
applications are excluded from the antigen to be used in the
vaccine composition of the present invention. In one embodiment,
one or more peptides selected from the group consisting of HER2/neu
E75 peptide for cancer vaccine applications and a modified HER2/neu
E75 peptide for cancer vaccine applications are excluded from the
antigen to be used in the vaccine composition of the present
invention.
[0024] The term "survivin-2B peptide" as used herein means a
peptide derived from a cancer gene product, survivin, consisting of
Ala Tyr Ala Cys Asn Thr Ser Thr Leu (SEQ NO: 1).
[0025] The term "GPC3 peptide" as used herein means a peptide
derived from a cancer gene product, GPC3, consisting of Glu Tyr Ile
Leu Ser Leu Glu Glu Leu (SEQ NO: 2).
[0026] The term "HER2/neu_A29 peptide" as used herein means an
HLA-A24-restricted peptide derived from a cancer gene product,
HER2/neu, consisting of Thr Tyr Leu Pro Thr Asn Ala Ser Leu (SEQ
NO: 3).
[0027] The term "MAGE3_A24 peptide" as used herein means an
HLA-A24-restricted peptide derived from a cancer gene product,
MAGE3, consisting of Ile Met Pro Lys Ala Gly Leu Leu Ile (SEQ NO:
4).
[0028] The term "IPEP87 peptide" as used herein means a peptide
derived from hepatitis C virus (HCV) protein, consisting of Asp Leu
Met Gly Tyr Ile Pro Ala Val (SEQ NO: 5).
[0029] The term "PR1 peptide" as used herein means a peptide
derived from a cancer gene product, proteinase-3, consisting of Val
Leu Gln Glu Leu Asn Val Thr Val (SEQ NO: 6).
[0030] The term "HER2/neu_A02 peptide" as used herein means an
HLA-A02-restricted peptide derived from a cancer gene product,
HER2/neu, consisting of Lys Val Phe Gly Ser Leu Ala Phe Val (SEQ
NO: 7).
[0031] The term "MAGE3_A02 peptide" as used herein means an
HLA-A02-restricted peptide derived from a cancer gene product,
MAGE3, consisting of Lys Val Ala Glu Ile Val His Phe Leu (SEQ NO:
8).
[0032] The term "HBVenv peptide" as used herein means a peptide
derived from hepatitis B virus (HBV) protein, consisting of Trp Leu
Ser Leu Leu Val Pro Phe Val (SEQ NO: 9).
[0033] The term "HER2/neu E75 peptide" as used herein means a
peptide derived from a product of cancer gene, HER2/neu (HER2
protein), consisting of Lys Ile Phe Gly Ser Leu Ala Phe Leu (SEQ
NO: 10).
[0034] The term "MUC1 peptide" as used herein means a peptide
derived from MUC1 protein, which is a glycoprotein highly expressed
on many cancer cells, consisting of Ser Thr Ala Pro Pro Val His Asn
Val (SEQ NO: 11).
[0035] The term "WT1 peptide" as used herein means a partial
peptide consisting of about 8 to about 15, preferably about 8 to
about 12, amino acids. The WT1 peptide is a peptide obtained by
fragmenting WT1 protein which is a product of a cancer gene, WT1
(Wilm's tumor), and include Db126 peptide and Db235 peptide (both
are described in Japanese Patent No. 4422903). In addition, a
partial peptide of WT1 product disclosed in WO 2000/06602, a
WR1-derived HLA-A26 binding cancer antigen peptide described in WO
2005/095598, an HLA-A* 3303-restricted WT1 peptide described in WO
2007/097358, and HLA-A* 1101-restricted WT1 peptide described in WO
2008/081701 are also included in the "WT1 peptide" of the present
invention.
[0036] The term "modified XX peptide" (XX is a name of arbitrary
peptide) as used herein means a modified peptide in which all or a
part of amino acids in a XX peptide are substituted or
modified.
[0037] Examples of the modified XX peptide include:
(a) a peptide including an amino acid sequence in which one to
several, for example, 1, 2, 3, 4 or 5 amino acids are substituted,
deleted or added in the amino acid sequence of the XX peptide; and
(b) a peptide including an amino acid sequence in which all or a
part of amino acids, for example, one to plural amino acids, such
as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are modified in the
amino acid sequence of the XX peptide.
[0038] Examples of the "modification" in the amino acid which may
occur in the modified XX peptide include, while not limited
thereto, aliphatic chain addition modification such as acetylation,
alkylation such as methylation, glycosylation, hydroxylation,
carboxylation, aldehydation, phosphorylation, sulfonylation,
formylation, modification by addition of an aliphatic chain such as
myristoylation, palmitoylation or stearoylation, octanoylation,
esterification, amidation, deamidation, modification by disulfide
bond formation such as cystine modification, glutathione
modification or thioglycolic acid modification, glycation,
ubiquitination, succinimide formation, glutamylation, prenylation,
and the like. The modified XX peptide may include a combination of
one or more amino acids substituted, deleted or added with one or
more amino acids modification.
[0039] In a preferable aspect, the antigen contained in the vaccine
composition for mucosal administration of the present invention is
a peptide selected from the group consisting of survivin-2B peptide
and/or modified survivin-2B peptide, GPC3 peptide and/or modified
GPC3 peptide, HER2/neu_A24 peptide and/or modified HER2/neu_A24
peptide, MAGE3_A24 peptide and/or modified MAGE3_A24 peptide,
IPEP87 peptide and/or modified IPEP87 peptide, PR1 peptide and/or
modified PR1 peptide, HER2/neu_A02 peptide and/or modified
HER2/neu_A02 peptide, MAGE3_A02 peptide and/or modified MAGE3_A02
peptide, HBVenv peptide and/or modified HBVenv peptide, and MUC1
peptide and/or modified MUC1 peptide. Alternatively, HER2/neu E75
peptide and/or modified HER2/neu E75 peptide may be used as the
antigen.
[0040] The peptides listed above can be in free form or in the form
of any pharmacologically acceptable salt thereof, for example, acid
salts (acetate, TFA salt, hydrochloride, sulfate, phosphate,
lactate, tartrate, maleate, fumarate, oxalate, hydrobromide,
succinate, nitrate, malate, citrate, oleate, palmitate, propionate,
formate, benzoate, picrate, benzenesulfonate, dodecylsulfate,
methanesulfonate, p-toluenesulfonate, glutarate, various amino acid
salts, and the like), metal salts (alkali metal salts (e.g. sodium
salt and potassium salt), alkaline earth metal salts (e.g. calcium
salt and magnesium salt), aluminum salts, and the like), and amine
salts (triethylamine salt, benzylamine salt, diethanolamine salt,
t-butylamine salt, dicyclohexylamine salt, arginine salt,
dimethylammonium salt, ammonium salt, and the like). The
pharmacologically acceptable salt is preferably acetate and a TFA
salt. The peptides described above, which can be used as the
antigen in the present invention, may be synthesized or produced by
a well-known method, followed by isolation and purification.
[0041] The term "cellular immunity induction promoter" as used
herein means any substance which can more enhance immune response
induced by an antigen, which is administered together with the
substance, as compared with the immune response induced by the
antigen without the substance. The cellular immunity induction
promoter may include substances specified in the specification of
the present invention, though it is not limited by the action
mechanism by which cellular immunity induction is promoted.
[0042] The term "TLR ligand" as used herein means a ligand of a
Toll-like receptor (TLR), and include, for example, ligands of TLR1
to TLR9. Examples of the TLR ligand include a ligand of a
heterodimer of TLR1 and TLR2 (TLR1/2 ligand), a ligand of a
heterodimer of TLR2 and TLR6 (TLR2/6 ligand), a TLR2 and Dectin 1
ligand, a TLR3 ligand, a TLR4 ligand, a TLR5 ligand, a TLR7 and/or
TLR8 ligand, a TLR9 ligand, and the like. Any of them can be used
as the cellular immunity induction promoter in the present
invention. In a preferable aspect of the present invention, the TLR
ligand is one selected from the group consisting of a TLR1/2
ligand, a TLR3 ligand, a TLR4 ligand, and a TLR7 and/or TLR8
ligand.
[0043] The term "TLR1/2 ligand" as used herein means a ligand of a
heterodimer of Toll-like receptor (TLR) 1 and Toll-like receptor
(TLR) 2. Examples include triacylated lipoprotein derived from a
cell wall of bacterium and a salt thereof, which may be an extract,
a product or a synthetic product, but is not limited thereto.
[0044] In a preferable aspect of the present invention, the TLR1/2
ligand is Pam.sub.3CSK.sub.4. Pam.sub.3CSK.sub.4 has the
formula:
##STR00001##
[0045] The term "TLR2 ligand" as used herein means TLR2 in the
heterodimer of Toll-like receptor (TLR) 1 and Toll-like receptor
(TLR) 2, TLR2 in the heterodimer of Toll-like receptor (TLR) 6 and
Toll-like receptor (TLR) 2, and a ligand acting with both of them.
The TLR2 ligand includes, but is not limited to, for example,
bacterial cell wall-derived lipoteichoic acid, and peptidoglycan
and a salt thereof, which may be an extract, a product or a
synthetic product. In a preferable aspect of the present invention,
the TLR2 ligand is peptidoglycan (PGN).
[0046] The term "TLR2 and Dectin 1 ligand" as used herein means a
ligand of Toll-like receptor (TLR) 2 and .beta.1,3-glucan receptor
(Dectin 1), and includes, for example, a .beta.1,3-glucan derived
from a cell wall of fungus, and a salt thereof, which may be an
extract, a product, or a synthetic product, but is not limited
thereto. In a preferable aspect of the present invention, the TLR2
and Dectin 1 ligand is Zymosan derived from a cell wall of
yeast.
[0047] The term "TLR3 ligand" as used herein means a ligand of
Toll-like receptor (TLR) 3. Examples include double stranded RNA
(dsRNA) derived from virus, and a salt thereof, which may be an
extract, a product, or a synthetic product, but is not limited
thereto. In a preferable aspect of the present invention, the TLR3
ligand is polyinosinic-polycytidylic acid (Poly(I:C)) which is a
synthetic product, and/or a salt thereof.
[0048] The term "TLR4 ligand" as used herein means a ligand of
Toll-like receptor (TLR) 4. Examples includes, but is not limited
to, lipopolysaccharide (LPS) derived from bacteria or plant, in
particular, lipid A derivatives such as monophosphoryl lipid A,
3-deacylated monophosphoryl lipid A (3D-MPL), OM 174, OM 294 DP or
OM 197 MP-Ac DP; alkyl glucosaminide phosphate (AGP), for example,
AGP disclosed in WO 9850399 or U.S. Pat. No. 6,303,347, or a salt
of AGP disclosed in U.S. Pat. No. 6,764,840, lipopolysaccharide
derived from Pantoea bacterium, glucopyranosyl lipid, and sodium
hyaluronate.
[0049] In a preferable aspect of the present invention, the TLR4
ligand is preferably a lipopolysaccharide derived from the genus
Acetobacter (for example, Acetobacter aceti, Acetobacter xylinum,
and Acetobacter orientalis), the genus Zymomonas (for example,
Zymomonas mobilis, and the like), the genus Xanthomonas (for
example, Xanthomonas campestris), the genus Enterobacter (for
example, Enterobacter cloacae), or the genus Pantoea (for example,
Pantoea agglomerans). It is possible to use the extract derived
from the lipopolysaccharide or purified lipopolysaccharide as it
is. In addition, for example, lipopolysaccharide (IP-PA1) derived
from Pantoea agglomerans is available from Funakoshi Corporation.
In a preferable aspect of the present invention, the TLR4 ligand is
lipopolysaccharide derived from Pantoea bacterium, glucopyranosyl
lipid and/or sodium hyaluronate.
[0050] The term "TLR7 and/or TLR8 ligand" as used herein means a
ligand of Toll-like receptor (TLR) 7 and/or TLR 8. It includes, but
is not limited to, for example, single stranded RNA, imiquimod,
resiquimod (R848), TLR7--II, and other compounds such as loxoribine
and bropirimine.
[0051] In a preferable aspect of the present invention, the TLR7
and/or TLR8 ligand is imiquimod. The imiquimod is
1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine of the
formula:
##STR00002##
whose characteristics and production method are described, for
example, in JP-A-7-505883 (Patent Document 4).
[0052] In another preferable aspect, the TLR7 and/or TLR8 ligand is
resiquimod. The resiquimod is
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinol-
in-a-ethanol of the formula:
##STR00003##
[0053] In another preferable aspect, the TLR7 and/or TLR8 ligand is
TLR7--II. The TLR7--II is represented by the formula:
##STR00004##
[0054] In another preferable aspect, the TLR7 and/or TLR8 ligand is
bropirimine. The bropirimine is represented by the formula:
##STR00005##
[0055] The term "TLR9 ligand" as used herein means a ligand of
Toll-like receptor (TLR) 9, and includes, for example, ODN 1826.
The TLR9 ligand used in the present invention may be an extract, a
product, or a synthetic product, but is not limited thereto. In a
preferable aspect of the present invention, the TLR9 ligand is ODN
1826.
[0056] ODN 1826 is oligodeoxynucleotide consisting of the following
sequence (SEQ NO: 12):
TABLE-US-00001 5'-tccatgacgttcctgacgtt-3'
[0057] The term "TLR2/6 ligand" as used herein means a ligand of a
heterodimer of Toll-like receptor (TLR) 2 and Toll-like receptor
(TLR) 6, and includes, for example, a diacylated lipoprotein
derived from a cell wall of mycoplasma, and a salt thereof, which
may be an extract, a product, or a synthetic product, but is not
limited thereto. In a preferable aspect of the present invention,
the TLR2/6 ligand is Pam.sub.2CSK.sub.4, MALP-2 and/or FSL-1.
[0058] PamCSK.sub.4 is represented by the following formula:
##STR00006##
[0059] FSL-1 is represented by the following formula:
##STR00007##
[0060] The term "TLR5 ligand" as used herein means a ligand of
Toll-like receptor (TLR) 5, and includes, for example, flagellin.
The TLR5 ligand used in the present invention may be an extract, a
product, or a synthetic product, but is not limited thereto. In a
preferable aspect of the present invention, the TLR5 ligand is
flagellin.
[0061] The Toll-like receptor (TLR) is a family of type I
transmembrane proteins, which initiates a congenital immune
response in a specific cytokine, a specific chemokine and a growth
factor participate, by in vivo activation thereof. All TLRs can
activate a certain intracellular signal transmission molecule, for
example, a nuclear factor KB (NF-KB) or a mitogen-activated protein
kinase (MAP kinase) or the like, while a specific population of a
cytokine and a chemokine which are released seems to be inherent in
each TLR. TLRs 3, 7, 8 and 9 include a subfamily of TLR which is
present in an endosome fraction or a lysosome fraction of an immune
cell (e.g. dendritic cells and monocytes). Specifically, TLR3 is
expressed by a wide range of cells such as a dendritic cell and a
fibroblast; TLR7 is expressed by a plasma-cell like dendritic cell
and is expressed by a monocyte to a lesser extent; TLR8 is
expressed by a monocyte, as well as a monocyte-derived dendritic
cell and a myeloid dendritic cell; and TLR9 is expressed by a
plasma-cell like dendritic cell. This subfamily mediates
recognition of microorganism nucleic acid (single stranded RNA,
double stranded RNA, single stranded DNA, and the like). Agonists
of TLR3, TLR7 and/or TLR8, or TLR9 stimulate production of various
inflammatory cytokines (which include, for example, interleukin-6,
interleukin-12, TNF-.alpha., and interferon-.gamma.). Such agonists
also promote increase in expression of a co-stimulator molecule
(for example, CD40, CD80, and CD86), a major histocompatibility
complex molecule, and a chemokine receptor. Type I interferons
(IFN-.alpha. and IFN-.beta.) are also produced by a cell upon
activation with TLR7 and/or TLR8 agonists.
[0062] The term "cyclic dinucleotide" as used herein means a
molecule in which two OH groups of a sugar part of two nucleotides
produce an ester for such same phosphoric acid molecule and thereby
nucleotides are cyclized and an analogs thereof, and includes, for
example, cyclic diAMP (c-di-AMP), cyclic diGMP (c-di-GMP), c-dGpGp,
c-dGpdGp, c-GpAp, c-GpCp, c-GpUp, and the like, but is not limited
thereto. The cyclic dinucleotide activates a dendritic cell or a T
cell. Further examples of the cyclic dinucleotide, use thereof as
an adjuvant, and a process for production thereof are described in
JP-A-2007-529531 (Patent Document 5). In a preferable aspect of the
present invention, the cyclic dinucleotide is cyclic diGMP and/or
cyclic diAMP. The cyclic diGMP has the formula:
##STR00008##
and a process for synthesis thereof is described in Kawai et al.,
Nucleic Acids Research Supp1.3:103-4.
[0063] The terms "helper peptide" as used herein means any peptide
which activates a helper T cell, and includes, for example,
tubercle bacillus-derived helper peptide, measles virus-derived
helper peptide, hepatitis B virus-derived helper peptide, hepatitis
C virus-derived helper peptide, Chlamydia trachomatis-derived
helper peptide, Plasmodium falciparum sporozoite-derived helper
peptide, keyhole limpet haemocyanin-derived helper peptide, tetanus
toxin-derived helper peptide, pertussis toxin-derived helper
peptide, diphtheria toxin-derived helper peptide, cancer
cell-derived helper peptide (for example, IMA-MMP-001 helper
peptide, CEA-006 helper peptide, MMP-001 helper peptide, TGFBI-004
helper peptide, HER-2/neu (aa776-790) helper peptide, AE36 helper
peptide, AE37 helper peptide, MET-005 helper peptide, and BIR-002
helper peptide), and universal helper analogs (for example,
PADRE).
[0064] In the present invention, in place of or in combination with
the helper peptide described above, a peptide in which all or a
part of the amino acids in the helper peptide are modified by
substitution, modification or the like (hereinafter, referred to as
a "modified helper peptide") can also be used.
[0065] Examples of the modified helper peptide include:
(a) a peptide consisting of an amino acid sequence in which one to
several, for example, 1, 2, 3, 4 or 5 amino acids are substituted,
deleted, or added in the amino acid sequence of the original helper
peptide; and (b) a peptide consisting of an amino acid sequence in
which all or a part of amino acids, for example, 1 to several,
e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino
acids are modified in the amino acid sequence of the original
helper peptide.
[0066] Examples of the "modification" in the amino acid which may
occur in the modified helper peptide include, but is not limited
thereto, aliphatic chain addition modification such as acetylation,
alkylation such as methylation, glycosylation, hydroxylation,
carboxylation, aldehydation, phosphorylation, sulfonylation,
formylation, addition of fatty acid such as myristoylation,
palmitoylation, and stearoylation, octanoylation, esterification,
amidation, deamidation, modification by disulfide bond formation
such as cystine modification, glutathione modification or
thioglycolic acid modification, glycation, ubiquitination,
succinimide formation, glutamylation, prenylation, and the like.
The modified helper peptide may include a combination of
substitution, deletion or addition of one or more amino acids.
[0067] In a preferable aspect of the present invention, the helper
peptide consists of 10 to 18 amino acids, preferably 12 to 16 amino
acids, more preferably 13 to 15 amino acids. In a preferable aspect
of the present invention, the helper peptide is Peptide-25,
modified Peptide-25, or PADRE. One example of the modified
Peptide-25 is Peptide-25B. The Peptide-25 is a peptide of 15 amino
acids consisting of Phe Gln Asp Ala Tyr Asn Ala Ala Gly Gly His Asn
Ala Val Phe (SEQ NO: 13) which corresponds to amino acid residues
240 to 254 of Ag85B which is one of the major proteins secreted by
human tubercle bacillus (Mycobacterium tuberculosis). The
Peptide-25B is one example of the modified Peptide-25 obtained by
modification of a part of amino acids in the Peptide-25 for
increasing in immunostimulation effect thereof, and is a peptide of
15 amino acids consisting of Phe Gln Asp Ala Tyr Asn Ala Val His
Ala Ala His Ala Val Phe (SEQ NO: 14). PADRE is a peptide of 13
amino acids, consisting of D-Ala Lys cyclohexyl-Ala Val Ala Ala Trp
Thr Leu Lys Ala Ala D-Ala (SEQ NO: 15).
[0068] The term "immunomodulatory small molecule drug" as used
herein means a substance which activates or inhibits immune cells
such as a T cell, an NK cell and a macrophage, provided that the
TLR ligand, the cyclic dinucleotide and the helper peptide
described above are excluded. Examples of the immunomodulatory
small molecule drug include bestatin, pidotimod, levamisole,
golotimod, forphenicinol, and their derivatives, and
pharmacologically acceptable salts thereof. For example, the
pharmacologically acceptable salt of levamisole includes levamisole
hydrochloride and the like.
[0069] Bestatin is represented by the formula:
##STR00009##
[0070] Pidotimod is represented by the formula:
##STR00010##
[0071] Levamisole hydrochloride is represented by the formula:
##STR00011##
[0072] In the present invention, the immunomodulatory small
molecule drug is usually a compound having a molecular weight of
less than 1000, preferably less than 500. In a preferable aspect of
the present invention, the immunomodulatory small molecule drug is
one or more compounds selected from the group consisting of
bestatin, pidotimod and levamisole hydrochloride.
[0073] In one aspect of the present invention, it has been found
that a TLR ligand, a cyclic dinucleotide, a helper peptide, or an
immunomodulatory small molecule drug is preferable for the cellular
immunity induction, when a desired antigen is mucosally
administered. Accordingly, in one aspect, the cellular immunity
induction promoter of the present invention comprises one or more
substances selected from them. In a particularly preferable aspect
of the present invention, the cellular immunity induction promoter
is a combination of one or more substances selected from a TLR
ligand, a cyclic dinucleotide and an immunomodulatory small
molecule drug, with a helper peptide. Various methods have been
developed as a method for quantitatively measuring the cellular
immunity induction, and one or more of them, for example, the
ELISPOT method, described in Examples, may be used.
[0074] In a preferable aspect, the cellular immunity induction
promoter contained in the vaccine composition for mucosal
administration of the present invention is one or more members
selected from the group consisting of a TLR ligand, a cyclic
dinucleotide, a helper peptide and an immunomodulatory small
molecule drug, more preferably is a combination of one or more
substances selected from the group consisting of a TLR ligand, a
cyclic dinucleotide and an immunomodulatory small molecule drug,
with a helper peptide. In a particularly preferable aspect, the
cellular immunity induction promoter is a combination of one or
more substances selected from the group consisting of a TLR1/2
ligand, a TLR3 ligand, a TLR4 ligand, a TLR7 and/or TLR8 ligand, a
cyclic diGMP and levamisole hydrochloride, with a helper
peptide.
[0075] The term "non-invasive administration" as used herein means
an administration without positively providing physical irritation
and/or chemical irritation, preferably without physical irritation
to the mucous membrane (for example, a treatment of releasing a
mucous membrane, a treatment of giving damage to a mucous membrane,
or a treatment of perforating a mucous membrane).
[0076] The term "cancer" as used herein means a cancer associated
with abnormal expression of a cancer gene, for example, a cancer
with over-expression of cancer gene, for example, hematopoietic
tumor or solid cancer. Examples of the cancer gene include survivin
gene, GPC3 gene, HER2/neu gene, MAGE3 gene, MAGE A1 gene, MAGE
A3/A6 gene, MAGE A4 gene, MAGE12 gene, proteinase-3 gene, AFP gene,
CA-125 gene, CD44 gene, CEA gene, c-Kit gene, c-met gene, c-myc
gene, L-myc gene, COX2 gene, CyclinD1 gene, Cytokeratin-7 gene,
Cytokeratin-19 gene, Cytokeratin-20 gene, E2F1 gene, E2F3 gene,
EGFR gene, Gli1 gene, hCG.beta. gene, HIF-1.alpha. gene, HnRNP
A2/B1 gene, hTERT gene, MDM gene, MDR-1 gene, MMP-2 gene, MMP-9
gene, Muc-1 gene, Muc-4 gene, Muc-7 gene, NSE gene, ProGRP gene,
PSA gene, RCAS1 gene, SCC gene, Thymoglobulin gene, VEGF-A gene,
VEGF-A gene, and the like. The cancer associated with abnormal
expression of survivin gene includes malignant lymphoma, bladder
cancer, lung cancer, and colon cancer, but is not limited thereto.
The cancer associated with abnormal expression of GPC3 gene
includes liver cancer, bile duct cancer, and gastric cancer, but is
not limited thereto. The cancer associated with abnormal expression
of HER2/neu gene includes breast cancer, gastric cancer, ovarian
cancer, uterine cancer, bladder cancer, non-small-cell lung cancer,
prostate cancer, and the like, but is not limited thereto. The
cancer associated with abnormal expression of MAGE3 gene includes
melanoma, lung cancer, head and neck cancer, bladder cancer,
gastric cancer, esophageal cancer, liver cancer, and the like, but
is not limited thereto. The cancer associated with abnormal
expression of proteinase-3 gene includes acute myelogenous
leukemia, pancreatic cancer, and the like, but is not limited
thereto.
[0077] The terms "abnormal expression of a gene" as used herein
means that a gene expression level in cells is increased or
decreased remarkably, for example two times or more, or four times
or more, as compared with other cells of the same tissue. The term
"over-expression" means that the abnormal expression is an increase
in the expression level. The expression level of a gene can be
easily measured using any method well-known in the art.
[0078] The term "subject" as used herein means any animal whose
immune response can be induced by the administration of the vaccine
composition for mucosal administration in a practical stage, and
includes typically a mammal including human, mouse, rat, dog, cat,
rabbit, horse, cow, sheep, pig, goat, monkey, and chimpanzee. A
particularly preferable subject is human,
[0079] The term "model animal for immunological evaluation" as used
herein means a model animal for evaluating the immunity induction
property of the vaccine composition for mucosal administration,
specifically a model animal for evaluating the cellular immunity
induction level. In view of compatibility between the antigen in
the vaccine composition and MHC class 1 molecule in an animal, an
animal in which cellular immunity induction caused by the antigen
in the vaccine composition can be evaluated is used as the model
animal for immunological evaluation. For example, in case of a
vaccine composition containing HLA-A* type 24 MHC-restricted class
1 peptide, the property is evaluated with BALB/c mice. In case of a
vaccine composition containing HLA-A* type 02 MHC-restricted
peptide, the property is evaluated with gene modified mouse in
which cellular immunity induction by HLA-A* type 02 MHC-restricted
peptide can be evaluated. In case of a vaccine composition
containing other HLA type MHC-restricted peptide, the property is
evaluated in an animal in which cellular immunity induction by the
HLA type MHC-restricted peptide can be evaluated. In case of a
vaccine composition containing a protein antigen, the property is
evaluated in an animal having MHC compatible with a class 1 epitope
which is intended to provide immunity induction among class 1
epitopes contained in the amino acid sequence of the protein
antigen.
[0080] The term "cancer antigen" as used herein means a substance
such as protein or peptide capable of specifically expressing tumor
cells or cancer cells, and of inducing an immune response.
[0081] The term "cancer antigen peptide" as used herein means a
partial peptide derived from a cancer antigen protein, which can
induce an immune response. The cancer antigen peptide is usually
generated by decomposition of a cancer antigen protein, which is a
cancer gene product, in the cancer cell, and is represented on the
surface of the cancer cell by MHC class I molecule. The cancer
antigen peptide used in a cancer vaccine formulation may be an
endogenous cancer antigen peptide which is isolated and purified
from a cancer cell, or may be a synthetic peptide having the same
amino acid sequence as that of the endogenous cancer antigen
peptide. In a preferable aspect of the present invention, the
cancer antigen peptide to be used for inducing immunity is, for
example, an endogenous or synthetic cancer antigen peptide selected
from the group consisting of survivin-2B peptide and/or modified
survivin-2B peptide, GPC3 peptide and/or modified GPC3 peptide,
HER2/neu_A24 peptide and/or modified HER2/neu_A24 peptide,
MAGE3_A24 peptide and/or modified MAGE3_A24 peptide, PR1 peptide
and/or modified PR1 peptide, HER2/neu_A02 peptide and/or modified
HER2/neu_A02 peptide, MAGE3_A02 peptide and/or modified MAGE3_A02
peptide, and MUC1 peptide and/or modified MUC1 peptide.
[0082] The term "virus antigen" as used herein means a substance
derived from a virus or a constituent component thereof, or a
substance derived therefrom, which can induce an immune response.
Accordingly, a viral disease can be treated or prevented by
mucosally administering the virus antigen, preferably together with
a cellular immunity induction promoter, to a subject. In a
preferable aspect of the present invention, for example, a peptide
selected from the group consisting of IPEP87 peptide and/or
modified IPEP87 peptide, and HBVenv peptide and/or modified HBVenv
peptide can be used as the virus antigen.
[0083] The term "viral disease" as used herein means a disease
caused by infection and proliferation of virus. Examples include
hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E,
cervical cancer, condyloma acuminatum, HIV infection, Chlamydia
infection, herpes simplex, and the like.
II. VACCINE COMPOSITION FOR MUCOSAL ADMINISTRATION
[0084] The vaccine composition for mucosal administration of the
present invention exhibits a high cellular immunity induction
effect by the mucosal administration of various antigens to a
subject.
[0085] The term the "composition for mucosal administration" as
used herein may be any formulation usually used for mucosal
administration such as a sublingual, transnasal, buccal, rectal or
vaginal administration, a semi-solid formulation such as a gel
formulation (jelly formulation), a cream formulation, an ointment,
or a plaster; a liquid formulation; a solid formulation such as a
powder formulation, a fine granule formulation, a granule
formulation, a film formulation, a tablet formulation, or an
orally-disintegrating tablet formulation; a spray formulation for a
mucous membrane such as an aerosol formulation; or an inhalating
formulation. The classification, definition, characteristics, and
production methods thereof are well-known in the art, and see, for
example, the Japanese Pharmacopoeia, the 16th edition.
[0086] As a solvent for the liquid formulation, for example, an
appropriate amount of a solvent such as water, ethanol, glycerol or
propylene glycol can be used, and the components can be dispersed
or dissolved in the solvent to prepare the liquid formulation.
[0087] Examples of the base material for the gel formulation (jelly
formulation) to be used as hydrogel base are a carboxyvinyl
polymer, a gel base, a fat-free ointment, polyvinyl pyrrolidone,
polyvinyl alcohol, sodium polyacrylate, carboxymethylcellulose,
starch, xanthan gum, karaya gum, sodium alginate, methylcellulose,
hydroxypropyl cellulose, hydroxypropyl methylcellulose phthalate
(HPMCP), cellulose acetate phthalate (CAP),
carboxymethylethylcellulose (CMEC), ethylcellulose,
hydroxyethylcellulose, hydroxypropyl methylcellulose, a
carboxyvinyl polymer, tragacanth, gum arabic, tara gum, tamarind
seed gum, psyllium seed gum, agar, gellan gum, glucomannan, locust
bean gum, guar gum, carrageenan, dextrin, dextran, amylose,
carboxymethylcellulose potassium, carboxymethylcellulose sodium,
carboxymethylcellulose calcium, pullulan, chitosan, carboxymethyl
starch sodium, Plantago testa, galactomannan, Eudragit, casein,
alkyl ester of alginic acid, gelatin, and polyethylene glycol.
These base materials can be dissolved in the solvent to prepare a
gel formulation having fluidity or formability. The solvent is
preferably water, and glycerol and propylene glycol can also be
used.
[0088] Examples of the base material for the cream formulation
include water/oil base materials such as a hydrophilic ointment and
vanishing cream; and oil/water base materials such as hydrophilic
vaseline, purified lanolin, Aquahole, Eucerin, Neocerin, hydrous
lanolin, cold cream, and hydrophilic plastibase. These base
materials are added to a fatty solvent or water, and the mixture is
stirred at a high speed using a homogenizer to prepare the cream
formulation.
[0089] Examples of the base material for the film formulation
include polyvinyl pyrrolidone, polyvinyl alcohol, sodium
polyacrylate, carboxymethyl cellulose, starch, xanthan gum, karaya
gum, sodium alginate, methylcellulose, a carboxyvinyl polymer,
agar, hydroxypropyl cellulose, hydroxypropyl methylcellulose
phthalate (HPMCP), cellulose acetate phthalate (CAP),
carboxymethylethylcellulose (CMEC), ethylcellulose,
hydroxyethylcellulose, hydroxypropyl methylcellulose, a
carboxyvinyl polymer, tragacanth, gum arabic, locust bean gum, guar
gum, carrageenan, dextrin, dextran, amylose, carboxymethylcellulose
potassium, carboxymethylcellulose sodium, carboxymethylcellulose
calcium, pullulan, chitosan, carboxymethyl starch sodium, Plantago
testa, galactomannan, an aminoalkyl methacrylate copolymer E, an
aminoalkyl methacrylate copolymer RS, methacrylic acid copolymer L,
methacrylic acid copolymer LD, methacrylic acid copolymer S, a
methylacrylate-methacrylic acid-methyl methacrylate copolymer, an
ethyl acrylate-methyl methacrylate copolymer, polyvinyl acetal
diethyl aminoacetate, casein, and an alginic acid alkyl ester.
These base materials are dissolved in water or a polar organic
solvent such as ethanol, the mixture is applied to form a thin
film, and the film is dried, whereby the film formulation can be
prepared. In one preferable aspect, the vaccine composition for
mucosal administration of the present invention is in the form of
film formulation.
[0090] For preparing the powder formulation, fine granule
formulation, granule formulation or tablet formulation, there are
used an excipient such as lactose, corn starch or crystalline
cellulose, and a binding agent such as hydroxypropyl cellulose and
gum arabic, and these additives are mixed in an appropriate amount
of a solvent such as water or ethanoland stirred, and followed by
granulation, drying and tableting steps. If necessary, a lubricant
such as magnesium stearate and a coating agent such as
hydroxypropyl cellulose or sucrose may be added.
[0091] Examples of the base material for the orally-disintegrating
tablet (lyophilization type) include polysaccharides such as
gelatin and pullulan. As a forming agent, mannitol, trehalose,
sorbitol, glycine, and the like may be used. These additives are
dissolved in water, and the mixture is lyophilized after
dispensation, whereby the orally-disintegrating tablet
(lyophilization-type) can be prepared. In one preferable aspect,
the vaccine composition for mucosal administration of the present
invention is in the form of orally-disintegrating tablet.
[0092] Examples of the aerosol formulation include a liquid
formulation, a gel formulation having a high fluidity, a cream
formulation, a fine powder such as a powder formulation, as the
content thereof. They are dispersed in gas in the form of solid or
liquid fine particles using a spraying device, whereby it can be
efficiently administered to an administration site such as an oral
mucous membrane or nasal mucous membrane.
[0093] A ratio between the antigen and the cellular immunity
induction promoter in the composition of the present invention is
not particularly limited. In one aspect, the composition of the
present invention preferably includes the desired antigen in an
amount of 0.01 to 40% by weight, more preferably 0.1 to 30% by
weight, based on the total weight of the composition. In one
aspect, the composition of the present invention preferably
contains the cellular immunity induction promoter in an amount of
0.001 to 30% by weight, more preferably 0.01 to 20% by weight,
based on the total weight of the composition.
[0094] The composition of the present invention, if necessary, may
include other additives. The additives can be selected, for
example, from an isotonizing agent, an antiseptic/antimicrobial
agent, an antioxidant, a solubilizer, a solubilizing aid, a
suspending agent, a filler, a pH-controlling agent, a stabilizer,
an absorption promoter, a releasing speed controller, a coloring
agent, a plasticizer, a cross-linking agent, an adhesive, and a
mixture of two or more thereof, which are selected appropriately in
view of the compatibility with the main component of the base, the
antigen, and the cellular immunity induction promoter, the desired
dosage regimen, and the like.
[0095] The composition of the present invention can further contain
a pharmacologically acceptable acid or a pharmacologically
acceptable salt thereof, which is a second cellular immunity
induction promoter, to improve immunity induction promoting
effect.
[0096] The "pharmacologically acceptable acid" as used herein,
which can be contained in the composition of the present invention,
means an acid which does not provide a harmful effect to a subject
to be administered, and does not extinguish the pharmacological
activity in the component in the composition. In a preferable
aspect of the present invention, the pharmacologically acceptable
acid is an organic acid; more preferably an organic compound
containing carboxyl group or an organic compound containing
sulfonate group; more preferably a saturated or unsaturated
straight or branched fatty acid having a saturated straight moiety
having 8 to 20 carbon atoms, lactic acid, malic acid, salicylic
acid, maleic acid, citric acid, or an organic compound containing
sulfonate group; more preferably a saturated or unsaturated
straight or branched fatty acid having a saturated straight moiety
having 8 to 16 carbon atoms, lactic acid, malic acid, salicylic
acid, maleic acid, citric acid, or an organic compound containing
sulfonate group; more preferably a fatty acid selected from the
group consisting of decanoic acid, lauric acid, myristic acid,
isostearic acid, palmitic acid, stearic acid and oleic acid, or
lactic acid, salicylic acid, citric acid or methanesulfonic
acid.
[0097] The "pharmacologically acceptable salt" as used herein,
which can be contained in the composition of the present invention,
means a salt which does not provide a harmful effect to a subject
to be administered, and does not extinguish the pharmacological
activity in the component in the composition.
[0098] The salt includes, but is not limited to, inorganic acid
salts (e.g., hydrochloride and phosphate), organic acid salts
(e.g., acetate, phthalate, and TFA salt), metal salts (alkali metal
salts (e.g., sodium salt and potassium salt), alkaline earth salts
(for example, calcium salt and magnesium salt), aluminum, salts,
and the like), and amine salts (triethylamine salt, benzylamine
salt, diethanolamine salt, t-butylamine salt, dicyclohexylamine
salt, arginine salt, dimethylammonium salt, ammonium salt, and the
like).
[0099] The therapeutically effective amount of the antigen can
widely vary depending on the severity of a disease, the age and
relative health of a subject, and other known factors, and in
general, a daily dose of about 0.1 .mu.g to 1 g/kg body weight can
provide a satisfactory result. The cellular immunity induction
promoter is administered simultaneously or sequentially when the
antigen is administered, preferably simultaneously. The effective
amount of the cellular immunity induction promoter can widely vary
depending on the specific cellular immunity induction promoter to
be used, and the presence or absence of another cellular immunity
induction promoter, and an amount of 0.01 .mu.g to 1 g/kg body
weight can provide a satisfactory result. A daily dose can be
administered once, and it may be divided into two or more, for
example, two, three, four or five aliquots, and administered.
Although the interval between administrations is arbitrary
interval, and is appropriately selected depending on the state of a
patient, the severity of a disease, and whether the purpose is
therapeutic or preventive, from, for example, once a day, once
every three days, once a week, once every two weeks, once a month,
once every three months, once every six months, once a year, or
more. In general, for the therapeutic purpose of a patient actually
suffering from a severe disease, the antigen is more frequently
administered in a higher dose, and for the preventive purpose of a
patient suffering from no disease, the antigen is fewer frequently
administered in a lower dose.
[0100] The present invention is illustrated more particularly and
specifically by the following Examples, but should not be construed
to be limited thereto.
EXAMPLES
Liquid Formulation for Sublingual Administration
[0101] Each liquid formulation having each composition shown in
Tables 1 to 9 below was produced and used as an administration
sample in mouse immunity experiments. Specifically, 20 parts by
weight of an additive (DMSO) and saline as a base material were
added to an antigen peptide, a cellular immunity induction
promoter, and, if desired, a pharmacologically acceptable acid in
amounts set forth in Tables 1 to 9 so that the total amount was 100
parts by weight, and the resultant was mixed to prepare a liquid
formulation for sublingual administration.
[0102] GPC3 peptide, survivin 2B peptide, HER2/neu_A24 peptide,
MAGE3_A24 peptide, IPEP87 peptide, HER2/neu E75 peptide, PR1
peptide, HER2/neu_A02 peptide, MAGE3_A02 peptide, HBVenv peptide,
and Peptide-25 were all chemically synthesized and HPLC-purified
before use. OVA protein was purchased from Sigma-Aldrich.
[0103] Imiquimod was purchased from Tokyo Chemical Industry Co.,
Ltd. Cyclic diGMP (c-di-GMP) and cyclic diAMP (c-di-AMP) were
purchased from Biolog Life Science Institute. Pam.sub.3CSK.sub.4
manufactured by InvivoGen, Inc., Poly(I:C) manufactured by
InvivoGen, Inc., Pantoea bacterium-derived lipopolysaccharide
manufactured by MARCO.PHI., glucopyranosyl lipid manufactured
by
[0104] InvivoGen, Inc. (MPLAs), resiquimod (R848) manufactured by
InvivoGen, Inc., and levamisole hydrochloride manufactured by MP
Biomedical Inc. were used.
Film Formulation
[0105] To 46 parts by weight of D-mannitol (manufactured by
Roquette Corporate) and 2.6 parts by weight of polyethylene glycol
400 (manufactured by Wako Pure Chemical Industries, Ltd.) was added
150 parts by weight of purified water, and the mixture was stirred
with ultrasonic waves. To the mixture were added 46 parts by weight
of hydroxypropyl cellulose (HPC-SSL manufactured by Nippon Soda
Co., Ltd.), 5 parts by weight of an antigen peptide (chemically
synthesized and HPLC-purified product), 0.3 parts by weight of
Peptide-25 (chemically synthesized and HPLC-purified product), and
0.1 parts by weight of a cellular immunity induction promoter other
than the helper peptide, and the resultant was thoroughly stirred
and mixed. The solution was added dropwise in an amount of 1/100
(2.5 parts by weight) to a polyethylene terephthalate release film,
which was air-dried and then dried under reduced pressure to
provide 1 part by weight of a film formulation.
Orally-Disintegrating Tablet
[0106] To 20 parts by weight of gelatin (water-soluble gelatin CSF
manufactured by Nippi Inc.) and 74.6 parts by weight of D-mannitol
was added 500 parts by weight of purified water, and the components
were dissolved with stirring. To the solution were added 5 parts by
weight of an antigen peptide (chemically synthesized and
HPLC-purified product), 0.3 parts by weight of Peptide-25
(chemically synthesized and HPLC-purified product), and 0.1 parts
by weight of a cellular immunity induction promoter other than the
helper peptide, and they were dissolved. The solution was dispensed
into formed aluminum containers, which were subjected to a
lyophilization treatment overnight to provide an
orally-disintegrating tablet. In mouse immunity experiments, the
orally-disintegrating tablet was pulverized, and 10 mg of the
tablet powder was weighed and used.
Mouse Immunity Experiment 1 (Sublingual Administration)
[0107] Mouse immunity experiments for the sublingual administration
liquid formulation, film formulation and orally-disintegrating
tablet were performed. The experiments were performed in accordance
with the ELISPOT method. Specifically, in a case where the
administration was performed once, the liquid formulation, the film
formulation or the orally-disintegrating tablet was administered
sublingually to anesthetized mice, and the mice were kept for 2
minutes as they were, and were fed for 6 days. In a case where the
administration was performed twice, the same procedure as above was
repeated after 6 days of the first administration. After 6 days
from the final administration, the spleen was isolated, and the
antigen-specific cellular immunity induction level was evaluated in
accordance with the ELISPOT method.
(ELISPOT Method)
[0108] A suspension of splenocytes was prepared from the spleen
isolated. Splenocytes (3.times.10.sup.6 cells/well) and an antigen
(100 .mu.M for an antigen peptide, 100 .mu.g/mL for an antigen
protein) were added to wells of an ELISPOT plate to which
anti-mouse IFN-.gamma. antibodies were immobilized, together with a
culture medium, and were co-cultured in culture conditions of
37.degree. C. and 5% CO.sub.2 for 20 hours, and the number of spots
of IFN-.gamma. productive cells (the number of
spots/3.times.10.sup.6 cells) was evaluated.
[0109] The results of the immunity experiments are shown in Tables
1 to 9 below, together with the mice used, the dosage, and the
number of administrations. The "genetically modified mice" in
Tables are genetically modified mice from which the cellular
immunity induction owing to HLA-A* 0201 MHC-restricted peptide can
be evaluated. For comparison, the results obtained from immunity
caused by injection formulations described below (Comparative
Examples 2 to 4 and 6 to 8) are also shown in each Table.
TABLE-US-00002 TABLE 1 Result of Composition immunization Dosage
Antigen Cellular immunity (ELISPOT average form Base peptide
induction promoter Acid Dose Administration Mouse number of spots)
Comparative Liquid Saline Survivin None None None 20 .mu.L twice
BALB/c 5 Example 1 formulation 2B (2.5) Example 1 Liquid Saline
Survivin LPS (0.1) None None 20 .mu.L twice BALB/c 25 formulation
2B (2.5) Example 2 Liquid Saline Survivin None PEPB None 20 .mu.L
twice BALB/c 15 formulation 2B (2.5) (0.3) Example 3 Liquid Saline
Survivin LPS (0.1) PEPB None 20 .mu.L twice BALB/c 49 formulation
2B (2.5) (0.3) Example 4 Liquid Saline Survivin LPS (0.1) PEPB MA
(0.05) 20 .mu.L twice BALB/c 80 formulation 2B (2.5) (0.3) Example
5 Liquid Saline Survivin LPS (0.1) PEPB Isostearic 20 .mu.L twice
BALB/c 72 formulation 2B (2.5) (0.3) acid (0.05) Example 6 Liquid
Saline Survivin LPS (0.1) PEPB Lactic acid 20 .mu.L twice BALB/c 76
formulation 2B (2.5) (0.3) (0.05) Example 7 Liquid Saline Survivin
LPS (0.1) PEPB Citric acid 20 .mu.L twice BALB/c 70 formulation 2B
(2.5) (0.3) (0.05) Example 8 Liquid Saline Survivin syn-MPL PEPB
None 20 .mu.L twice BALB/c 40 formulation 2B (2.5) (0.1) (0.3)
Example 9 Liquid Saline Survivin Imiquimod PEPB None 20 .mu.L twice
BALB/c 20 formulation 2B (2.5) (0.3) (0.3) Example 10 Liquid Saline
Survivin c-di-GMP None None 10 .mu.L once BALB/c 254 formulation 2B
(2.5) (0.2) Example 11 Liquid Saline Survivin c-di-GMP PEPB None 10
.mu.L once BALB/c 333 formulation 2B (2.5) (0.2) (0.3) Example 12
Liquid Saline Survivin Levamisole PEPB None 20 .mu.L twice BALB/c
16 formulation 2B (2.5) HCl (0.5) (0.3) Example 13 Film HPC/PEG/
Survivin LPS (0.1) PEPB None 10 mg twice BALB/c 53 formulation
mannitol 2B (5) (0.3) Example 14 Orally Gelatin/ Survivin LPS (0.1)
PEPB None 10 mg twice BALB/c 54 disintegrating mannitol 2B (5)
(0.3) tablet Comparative Subcutaneous Saline Survivin 2B Montanide
None 200 .mu.L once BALB/c 313 Example 2 injection (0.125) ISA51VG
(50) Numbers in ( ) are a blending ratio of each component (part by
weight). (The same applies to the following Tables.) HPC:
Hydroxypropyl cellulose PEG: Polyethylene glycol 400 LPS: Pantoea
bacterium-derived lipopolysaccharide (TLR4 ligand) Imiquimod: TLR7
and/or TLR8 ligand syn-MPL: synthetic monophosphoryl lipid A
(Glucopyranosyl lipid) (TLR4 ligand) c-di-GMP: cyclic diGMP (cyclic
dinucleotide) Levamisole HCl: Levamisole hydrochloride
(immunomodulatory small molecule drug) PEPB: Peptide-25B (SEQ No:
14) (helper peptide) MA: Myristic acid
TABLE-US-00003 TABLE 2 Result of Composition immunization Antigen
Cellular immunity (ELISPOT average Dosage form Base peptide
induction promoter Acid Dose Administration Mouse number of spots)
Comparative Liquid Saline MAGE3_A02 (5) None None None 20 .mu.L
twice Genetically 6 Example 9 formulation modified Example 15
Liquid Saline MAGE3_A02 (5) None PEP (0.3) None 20 .mu.L twice
Genetically 15 formulation modified Example 16 Liquid Saline
MAGE3_A02 (5) LPS (0.1) None None 20 .mu.L twice Genetically 40
formulation modified Example 17 Liquid Saline MAGE3_A02 (5) LPS
(0.1) PEP (0.3) None 20 .mu.L twice Genetically 69 formulation
modified Example 18 Liquid Saline MAGE3_A02 (5) c-di-GMP None None
10 .mu.L once Genetically 650 formulation (0.2) modified Example 19
Liquid Saline MAGE3_A02 (5) c-di-GMP PEP (0.3) None 10 .mu.L once
Genetically 892 formulation (0.2) modified PEP: Peptide-25 (SEQ NO:
13) (helper peptide)
TABLE-US-00004 TABLE 3 Composition Result of Cellular immunity
immunization Antigen induction (ELISPOT average Dosage form Base
peptide promoter Acid Dose Administration Mouse number of spots)
Example 20 Liquid Saline HER2/neu_A24 c-di-GMP PEPB None 10 .mu.L
once BALB/c 69 formulation (10) (0.2) (0.3)
TABLE-US-00005 TABLE 4 Result of immunization Composition (ELISPOT
Antigen Cellular immunity average Dosage form Base peptide
induction promoter Acid Dose Administration Mouse number of spots)
Comparative Liquid Saline IPEP87 None None None 20 .mu.L twice
Genetically 4 Example 10 formulation (10) modified Example 21
Liquid Saline IPEP87 None PEP (0.3) None 20 .mu.L twice Genetically
10 formulation (10) modified Example 22 Liquid Saline IPEP87
LPS(0.1) None None 20 .mu.L twice Genetically 25 formulation (10)
modified Example 23 Liquid Saline IPEP87 LPS(0.1) PEP (0.3) None 20
.mu.L twice Genetically 42 formulation (10) modified Example 24
Liquid Saline IPEP87 Imiquimod None None 20 .mu.L twice Genetically
132 formulation (10) (0.3) modified Example 25 Liquid Saline IPEP87
Imiquimod PEP (0.3) None 20 .mu.L twice Genetically 184 formulation
(10) (0.3) modified Example 26 Liquid Saline IPEP87 c-di-GMP None
None 10 .mu.L once Genetically 230 formulation (10) (0.2) modified
Example 27 Liquid Saline IPEP87 c-di-GMP PEP (0.3) None 10 .mu.L
once Genetically 287 formulation (10) (0.2) modified
TABLE-US-00006 TABLE 5 Result of Composition immunization Dosage
Antigen Cellular immunity Administ- (ELISPOT average form Base
peptide induction promoter Acid Dose ration Mouse number of spots)
Comparative Liquid Saline PR1(1.25) None None None 10 .mu.L once
Genetically 6 Example 11 formulation modified Example 28 Liquid
Saline PR1(1.25) c-di-GMP None None 10 .mu.L once Genetically 153
formulation (0.2) modified Example 29 Liquid Saline PR1(1.25)
c-di-GMP PEP(0.3) None 10 .mu.L once Genetically 244 formulation
(0.2) modified Comparative Subcutaneous Saline PR1 Montanide
ISA51VG None 200 .mu.L once Genetically 144 Example 3 injection
(0.125) (50) modified
TABLE-US-00007 TABLE 6 Result of Composition immunization Dosage
Antigen Cellular immunity Adminis- (ELISPOT average form Base
peptide induction promoter Acid Dose tration Mouse number of spots)
Comparative Liquid Saline HER2/neu_A02 None None None 20 .mu.L
twice Genetically 3 Example 12 formulation (1.25) modified Example
30 Liquid Saline HER2/neu_A02 LPS(0.1) None None 20 .mu.L twice
Genetically 15 formulation (1.25) modified Example 31 Liquid Saline
HER2/neu_A02 LPS(0.1) PEP(0.3) None 20 .mu.L twice Genetically 21
formulation (1.25) modified Example 32 Liquid Saline HER2/neu_A02
c-di-GMP None None 10 .mu.L once Genetically 560 formulation (1.25)
(0.2) modified Example 33 Liquid Saline HER2/neu_A02 c-di-GMP
PEP(0.3) None 10 .mu.L once Genetically 718 formulation (1.25)
(0.2) modified Comparative Subcutaneous Saline HER2/neu_A02
Montanide ISA51VG None 200 .mu.L once Genetically 93 Example 4
injection (0.125) (50) modified
TABLE-US-00008 TABLE 7 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Acid Dose tration Mouse number of spots)
Comparative Liquid Saline HBVenv None None None 10 .mu.L once
Genetically 6 Example 13 formulation (1.25) modified Example 34
Liquid Saline HBVenv c-di-GMP None None 10 .mu.L once Genetically
542 formulation (1.25) (0.2) modified Example 35 Liquid Saline
HBVenv c-di-GMP PEP(0.3) None 10 .mu.L once Genetically 787
formulation (1.25) (0.2) modified
TABLE-US-00009 TABLE 8 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Acid Dose tration Mouse number of spots)
Comparative Liquid Saline HER2/neu None None None 20 .mu.L twice
Genetically 3 Example 5 formulation E75(1.25) modified Example 36
Liquid Saline HER2/neu LPS(0.1) None None 20 .mu.L twice
Genetically 15 formulation E75(1.25) modified Example 37 Liquid
Saline HER2/neu None PEP None 20 .mu.L twice Genetically 11
formulation E75(1.25) (0.3) modified Example 38 Liquid Saline
HER2/neu LPS(0.1) PEP None 20 .mu.L twice Genetically 30
formulation E75(1.25) (0.3) modified Example 39 Liquid Saline
HER2/neu LPS(0.1) PEP MA(0.05) 20 .mu.L twice Genetically 41
formulation E75(1.25) (0.3) modified Example 40 Liquid Saline
HER2/neu LPS(0.1) PEP Isostearic 20 .mu.L twice Genetically 38
formulation E75(1.25) (0.3) acid modified (0.05) Example 41 Liquid
Saline HER2/neu LPS(0.1) PEP Lactic 20 .mu.L twice Genetically 37
formulation E75(1.25) (0.3) acid modified (0.05) Example 42 Liquid
Saline HER2/neu LPS(0.1) PEP Citric 20 .mu.L twice Genetically 38
formulation E75(1.25) (0.3) acid modified (0.05) Example 43 Liquid
Saline HER2/neu syn-MPL PEP None 20 .mu.L twice Genetically 27
formulation E75(1.25) (0.1) (0.3) modified Example 44 Liquid Saline
HER2/neu Imiquimod PEP None 20 .mu.L twice Genetically 89
formulation E75(1.25) (0.3) (0.3) modified Example 45 Liquid Saline
HER2/neu Resiquimod PEP None 20 .mu.L twice Genetically 177
formulation E75(1.25) (0.1) (0.3) modified Example 46 Liquid Saline
HER2/neu c-di-GMP None None 10 .mu.L once Genetically 523
formulation E75(1.25) (0.2) modified Example 47 Liquid Saline
HER2/neu c-di-GMP PEP None 10 .mu.L once Genetically 611
formulation E75(1.25) (0.2) (0.3) modified Example 48 Liquid Saline
HER2/neu Levamisole None None 20 .mu.L twice Genetically 45
formulation E75(1.25) HCl (0.5) modified Example 49 Liquid Saline
HER2/neu Levamisole PEP None 20 .mu.L twice Genetically 70
formulation E75(1.25) HCl (0.5) (0.3) modified Example 50 Liquid
Saline HER2/neu LPS(0.1) PADRE None 20 .mu.L twice Genetically 33
formulation E75(1.25) (0.3) modified Example 51 Film HPC/PEG/
HER2/neu LPS(0.1) PEP None 10 mg twice Genetically 32 formulation
mannitol E75(5) (0.3) modified Example 52 Orally Gelatin/ HER2/neu
LPS(0.1) PEP None 10 mg twice Genetically 33 disintegrating
mannitol E75(5) (0.3) modified tablet Comparative Subcutaneous
Saline HER2/neu Montanide ISA51VG None 200 .mu.L once Genetically
110 Example 6 injection E75(0.125) (50) modified Resiquimod: TLR7
and/or TLR8 ligand
TABLE-US-00010 TABLE 9 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Acid Dose tration Mouse number of spots)
Comparative Liquid Saline OVA protein None None None 20 .mu.L twice
BALB/c 9 Example 14 formulation (1.25) Example 53 Liquid Saline OVA
protein LPS(0.1) None None 20 .mu.L twice BALB/c 120 formulation
(1.25) Example 54 Liquid Saline OVA protein LPS(0.1) PEPB None 20
.mu.L twice BALB/c 142 formulation (1.25) (0.3) Example 55 Liquid
Saline OVA protein LPS(0.1) PEPB MA(0.05) 20 .mu.L twice BALB/c 405
formulation (1.25) (0.3) Example 56 Liquid Saline OVA protein
LPS(0.1) PEPB Isostearic 20 .mu.L twice BALB/c 380 formulation
(1.25) (0.3) acid (0.05) Example 57 Liquid Saline OVA protein
LPS(0.1) PEPB Lactic acid 20 .mu.L twice BALB/c 365 formulation
(1.25) (0.3) (0.05) Example 58 Liquid Saline OVA protein LPS(0.1)
PEPB Citric acid 20 .mu.L twice BALB/c 345 formulation (1.25) (0.3)
(0.05) Example 59 Liquid Saline OVA protein c-di-GMP None None 10
.mu.L once BALB/c 457 formulation (1.25) (0.1) Example 60 Liquid
Saline OVA protein c-di-GMP PEPB None 10 .mu.L once BALB/c 524
formulation (1.25) (0.1) (0.3) Example 61 Film HPC/PEG/ OVA protein
LPS(0.1) PEPB None 10 mg twice BALB/c 145 formulation mannitol (5)
(0.3) Example 62 Orally Gelatin/ OVA protein LPS(0.1) PEPB None 10
mg twice BALB/c 147 disintegrating mannitol (5) (0.3) tablet
Liquid Formulation for Transnasal Administration
[0110] Each liquid formulation having each composition shown in
Tables 10 to 19 below was produced, and used as an administration
sample in a mouse immunity experiment. Specifically, to an antigen
peptide and a cellular immunity induction promoter in amounts set
forth in Tables 10 to 19 were added 20 parts by weight of an
additive (DMSO) and saline as a base material so that the total
amount was 100 parts by weight, and the resultant was mixed to
prepare a liquid formulation for transnasal administration. The
antigen peptide and the cellular immunity induction promoter were
obtained from the same companies as in the case of the liquid
formulation for sublingual administration described above.
Mouse Immunity Experiment 2 (Transnasal Administration)
[0111] A mouse immunity experiment for the liquid formulation for
transnasal administration was performed. The experiment was
performed in accordance with the ELISPOT method. Specifically, in a
case where the administration was performed once, after mice were
anesthetized, liquid formulation was absorbed from nasal cavities,
and the mice were fed for 6 days. In a case where the
administration was performed twice, the same procedure as above was
repeated after 6 days from the first administration. After 6 days
from the final administration, the spleen was isolated, and the
antigen-specific cellular immunity induction level was evaluated in
accordance with the ELISPOT method. The ELISPOT method was
performed in the same manner as in mouse immunity experiment 1.
[0112] The results of the immunity experiment are shown in Table 10
to 19 below, together with the mice used, the dosage, and the
number of administration. The "genetically modified mice" in Tables
are genetically modified mice from which the cellular immunity
induction owing to HLA-A* 0201 MHC-restricted peptide can be
evaluated. For comparison, the results obtained from immunity
caused by injection formulations described below (Comparative
Examples 2 to 4 and 6 to 8) were described at the end of each
Table.
TABLE-US-00011 TABLE 10 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Dose tration Mouse number of spots)
Comparative Liquid Saline Survivin 2B None None 10 .mu.L twice
BALB/c 3 Example 15 formulation (2.5) Example 63 Liquid Saline
Survivin 2B LPS(0.1) None 10 .mu.L twice BALB/c 15 formulation
(2.5) Example 64 Liquid Saline Survivin 2B LPS(0.1) PEPB 10 .mu.L
twice BALB/c 20 formulation (2.5) (0.3) Example 65 Liquid Saline
Survivin 2B poly(I:C) PEPB 10 .mu.L twice BALB/c 18 formulation
(2.5) (0.1) (0.3) Example 66 Liquid Saline Survivin 2B c-di-GMP
PEPB 10 .mu.L once BALB/c 142 formulation (2.5) (0.2) (0.3)
Comparative Subcutaneous Saline Survivin 2B Montanide ISA51VG 200
.mu.L once BALB/c 313 Example 2 injection (0.125) (50) poly(I:C):
Polyinosinic-polycytidylic acid (TLR3 ligand)
TABLE-US-00012 TABLE 11 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Dose tration Mouse number of spots)
Comparative Liquid Saline MAGE3_A02 (5) None None 10 .mu.L twice
Genetically 6 Example 16 formulation modified Example 67 Liquid
Saline MAGE3_A02 (5) None PEP(0.3) 10 .mu.L twice Genetically 12
formulation modified Example 68 Liquid Saline MAGE3_A02 (5)
LPS(0.1) None 10 .mu.L twice Genetically 42 formulation modified
Example 69 Liquid Saline MAGE3_A02 (5) LPS(0.1) PEP(0.3) 10 .mu.L
twice Genetically 59 formulation modified Example 70 Liquid Saline
MAGE3_A02 (5) c-di-GMP PEP(0.3) 10 .mu.L once Genetically 1151
formulation (0.2) modified
TABLE-US-00013 TABLE 12 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Dose tration Mouse number of spots)
Example 71 Liquid Saline MAGE3_A24 c-di-GMP PEPB 10 .mu.L once
BALB/c 78 formulation (10) (0.2) (0.3) Comparative Subcutaneous
Saline MAGE3_A24 Montanide ISA51VG 200 .mu.L once BALB/c 56 Example
7 injection (0.125) (50)
TABLE-US-00014 TABLE 13 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Dose tration Mouse number of spots)
Example 72 Liquid Saline GPC3 c-di-GMP PEPB 10 .mu.L once BALB/c 13
formulation (10) (0.2) (0.3) Comparative Subcutaneous Saline GPC3
Montanide ISA51VG 200 .mu.L once BALB/c 10 Example 8 injection
(0.125) (50)
TABLE-US-00015 TABLE 14 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Dose tration Mouse number of spots)
Example 73 Liquid Saline HER2/neu_A24 c-di-GMP PEPB 10 .mu.L once
BALB/c 28 formulation (10) (0.2) (0.3)
TABLE-US-00016 TABLE 15 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Dose tration Mouse number of spots)
Comparative Liquid Saline IPEP87 None None 10 .mu.L twice
Genetically 6 Example 17 formulation (10) modified Example 74
Liquid Saline IPEP87 None PEP(0.3) 10 .mu.L twice Genetically 53
formulation (10) modified Example 75 Liquid Saline IPEP87 LPS(0.1)
None 10 .mu.L twice Genetically 187 formulation (10) modified
Example 76 Liquid Saline IPEP87 LPS(0.1) PEP(0.3) 10 .mu.L twice
Genetically 295 formulation (10) modified Example 77 Liquid Saline
IPEP87 Imiquimod PEP(0.3) 10 .mu.L twice Genetically 229
formulation (10) (0.3) modified Example 78 Liquid Saline IPEP87
c-di-GMP PEP(0.3) 10 .mu.L once Genetically 909 formulation (10)
(0.2) modified Example 79 Liquid Saline IPEP87 Levamisole HCl
PEP(0.3) 10 .mu.L twice Genetically 235 formulation (10) (0.5)
modified
TABLE-US-00017 TABLE 16 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Dose tration Mouse number of spots)
Comparative Liquid Saline PR1 (1.25) None None 10 .mu.L twice
Genetically 2 Example 18 formulation modified Example 80 Liquid
Saline PR1 (1.25) None PEP(0.3) 10 .mu.L twice Genetically 10
formulation modified Example 81 Liquid Saline PR1 (1.25) LPS(0.1)
None 10 .mu.L twice Genetically 15 formulation modified Example 82
Liquid Saline PR1 (1.25) LPS(0.1) PEP(0.3) 10 .mu.L twice
Genetically 26 formulation modified Example 83 Liquid Saline PR1
(1.25) c-di-GMP PEP(0.3) 10 .mu.L once Genetically 92 formulation
(0.2) modified Comparative Subcutaneous Saline PR1 (0.125)
Montanide ISA51VG 200 .mu.L once Genetically 144 Example 3
injection (50) modified
TABLE-US-00018 TABLE 17 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Dose tration Mouse number of spots)
Comparative Liquid Saline HER2/neu_A02 None None 10 .mu.L twice
Genetically 5 Example 19 formulation (1.25) modified Example 84
Liquid Saline HER2/neu_A02 None PEP(0.3) 10 .mu.L twice Genetically
13 formulation (1.25) modified Example 85 Liquid Saline
HER2/neu_A02 LPS(0.1) None 10 .mu.L twice Genetically 84
formulation (1.25) modified Example 86 Liquid Saline HER2/neu_A02
LPS(0.1) PEP(0.3) 10 .mu.L twice Genetically 114 formulation (1.25)
modified Example 87 Liquid Saline HER2/neu_A02 c-di-GMP PEP(0.3) 10
.mu.L once Genetically 678 formulation (1.25) (0.2) modified
Comparative Subcutaneous Saline HER2/neu_A02 Montanide ISA51VG 200
.mu.L once Genetically 93 Example 4 injection (0.125) (50)
modified
TABLE-US-00019 TABLE 18 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Dose tration Mouse number of spots)
Example 88 Liquid Saline HBVenv c-di-GMP PEP(0.3) 10 .mu.L once
Genetically 329 formulation (1.25) (0.2) modified
TABLE-US-00020 TABLE 19 Result of Composition immunization Antigen
Cellular immunity Adminis- (ELISPOT average Dosage form Base
peptide induction promoter Dose tration Mouse number of spots)
Comparative Liquid Saline HER2/neu E75 None None 10 .mu.L twice
Genetically 3 Example 20 formulation (1.25) modified Example 89
Liquid Saline HER2/neu E75 None PEP(0.3) 10 .mu.L twice Genetically
11 formulation (1.25) modified Example 90 Liquid Saline HER2/neu
E75 LPS(0.1) None 10 .mu.L twice Genetically 16 formulation (1.25)
modified Example 91 Liquid Saline HER2/neu E75 LPS(0.1) PEP(0.3) 10
.mu.L twice Genetically 31 formulation (1.25) modified Example 92
Liquid Saline HER2/neu E75 Pam3CSK4 PEP(0.3) 10 .mu.L twice
Genetically 69 formulation (1.25) (0.1) modified Example 93 Liquid
Saline HER2/neu E75 poly(I:C) PEP(0.3) 10 .mu.L twice Genetically
183 formulation (1.25) (0.1) modified Example 94 Liquid Saline
HER2/neu E75 c-di-GMP PEP(0.3) 10 .mu.L once Genetically 476
formulation (1.25) (0.2) modified Example 95 Liquid Saline HER2/neu
E75 LPS(0.1) PADRE 10 .mu.L twice Genetically 34 formulation (1.25)
(0.3) modified Comparative Subcutaneous Saline HER2/neu E75
Montanide ISA51VG 200 .mu.L once Genetically 110 Example 6
injection (0.125) (50) modified Pam3CSK4: TLR1/2 ligand
Subcutaneous Injection Formulation
[0113] Each subcutaneous injection formulation having each
composition shown in Table 20 below was produced, and used as an
administration sample in an immunity experiment. Specifically, to
an antigen peptide and an adjuvant, Montanide ISA51VG (Freund
Corporation), in amounts set forth in Table 20, were added 0.5
parts by weight of an additive (DMSO) and saline as a base material
so that the total amount was 100 parts by weight, and the resultant
was mixed to prepare an injection formulation. The antigen peptide
was obtained from the same company as in the case of the liquid
formulation for sublingual administration described above.
Mouse Immunity Experiment 3 (Subcutaneous Injection)
[0114] A mouse immunity experiment for the subcutaneous injection
formulation described above was performed. The experiment was
performed in accordance with the ELISPOT method. Specifically,
after 200 .mu.L of the formulation was subcutaneously
injection-administered to the back of a mouse, the mouse was raised
for 6 days. After 6 days from the day on which the administration
was performed, the spleen was extirpated, and the antigen-specific
cellular immunity induction level was evaluated in accordance with
the ELISPOT method. The number of administrations was once in every
case. The ELISPOT method was performed in the same manner as in
mouse immunity experiment 1.
[0115] The results of the immunity experiment are shown in Table 20
below, together with the mice used. The "genetically modified mice"
in Table are genetically modified mice from which the cellular
immunity induction owing to HLA-A* 0201 MHC-restricted peptide can
be evaluated.
TABLE-US-00021 TABLE 20 Composition Result of immunization Cellular
immunity (ELISPOT average number Base Antigen peptide induction
promoter Mouse of spots) Comparative Saline Survivin 2B (0.125)
Montanide ISA51VG(50) BALB/c 313 Example 2 Comparative Saline PR1
(0.125) Montanide ISA51VG(50) Genetically 144 Example 3 modified
Comparative Saline HER2/neu_A02 (0.125) Montanide ISA51VG(50)
Genetically 93 Example 4 modified Comparative Saline HER2/neu E75
(0.125) Montanide ISA51VG(50) Genetically 110 Example 6 modified
Comparative Saline MAGE3_A24 (0.125) Montanide ISA51VG(50) BALB/c
56 Example 7 Comparative Saline GPC3 (0.125) Montanide ISA51VG(50)
BALB/c 10 Example 8
[0116] In Tables 1-9, a vaccine composition for mucosal
administration comprising an antigen and a cellular immunity
induction promoter was administered in sublingual route, and an
efficacy of the cellular immunity induction promoter was
evaluated.
[0117] As a result, one or more cellular immunity induction
promoters selected from the group consisting of TLR ligand, a
cyclic dinucleotide, a helper peptide and an immunomodulatory small
molecule drug were effective.
[0118] Preferably, a cellular immunity induction promoter selected
from TLR4 ligand, TLR7 and/or TLR8 ligand, a cyclic dinucleotide, a
helper peptide, an immunomodulatory small molecule drug and a
combination of two or more of them was effective.
[0119] More preferably, TLR4 ligand, TLR7 and/or TLR8 ligand, a
cyclic dinucleotide, and a combination of these compounds with a
helper peptide were particularly effective.
[0120] From the viewpoint of safety, a sublingual administration is
preferable rather than a nasal administration. However, the strong
induction of immunity was also confirmed when a film formulation or
an orally-disintegrating tablet, which is a preferable form in view
of convenient administration and storage stability, was used.
[0121] In Tables 10-19, a vaccine composition for mucosal
administration comprising an antigen and a cellular immunity
induction promoter was administered in nasal route, and an efficacy
of the cellular immunity induction promoter was evaluated.
[0122] One or more cellular immunity induction promoters selected
from the group consisting of TLR ligand, a cyclic dinucleotide, a
helper peptide and an immunomodulatory small molecule drug were
effective.
[0123] Preferably, a cellular immunity induction promoter selected
from TLR1/2 ligand, TLR3 ligand, TLR4 ligand, TLR7 and/or TLR8
ligand, a cyclic dinucleotide, a helper peptide, an
immunomodulatory small molecule drug and a combination of two or
more of them was effective.
Sequence CWU 1
1
1719PRTHomo sapiens 1Ala Tyr Ala Cys Asn Thr Ser Thr Leu 1 5
29PRTHomo sapiens 2Glu Tyr Ile Leu Ser Leu Glu Glu Leu 1 5
39PRTHomo sapiens 3Thr Tyr Leu Pro Thr Asn Ala Ser Leu 1 5
49PRTHomo sapiens 4Ile Met Pro Lys Ala Gly Leu Leu Ile 1 5
59PRTHepatitis C virus 5Asp Leu Met Gly Tyr Ile Pro Ala Val 1 5
69PRTHomo sapiens 6Val Leu Gln Glu Leu Asn Val Thr Val 1 5
79PRTHomo sapiens 7Lys Val Phe Gly Ser Leu Ala Phe Val 1 5
89PRTHomo sapiens 8Lys Val Ala Glu Ile Val His Phe Leu 1 5
99PRTHepatitis B virus 9Trp Leu Ser Leu Leu Val Pro Phe Val 1 5
109PRTHomo sapiens 10Lys Ile Phe Gly Ser Leu Ala Phe Leu 1 5
119PRTHomo sapiens 11Ser Thr Ala Pro Pro Val His Asn Val 1 5
1220DNAUnknownDescription of Unknown Bacterial DNA sequence
12tccatgacgt tcctgacgtt 201315PRTMycobacterium tuberculosis 13Phe
Gln Asp Ala Tyr Asn Ala Ala Gly Gly His Asn Ala Val Phe 1 5 10 15
1415PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Phe Gln Asp Ala Tyr Asn Ala Val His Ala Ala His
Ala Val Phe 1 5 10 15 1513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 15Ala Lys Ala Val Ala Ala Trp
Thr Leu Lys Ala Ala Ala 1 5 10 165PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 16Ser Lys Lys Lys Lys 1 5
179PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Gly Asp Pro Lys His Pro Lys Ser Phe 1 5
* * * * *